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Bioprocess-III (Detailed Chromatography)

Prepared by Mohd. Tayyab and


Nimish Gopal, IIT Roorkee

Anion exchange chromatography


(AEC)

Anion exchange chromatography

Beads are +vely charged


Eg DEAE-C (Diethylaminoethyl cellulose) matirx
-vely charged proteins bind to beads
+vely charged proteins will elute out
Add buffer to elute unbound proteins
Bound protein are eluted by changing pH or by passing a
gradually increasing linear salt gradient (NaCl high
concentration)
Generally used for acidic proteins
Acidic proteins have low pI
Why for acidic? If you want to use it for basic .very high pH
may denature protein.lose its activity

Matrix or Resin
Matrix Is Support
Covalently attached functional group (charged)
Matrix, polymeric, porous beads
Cellulose
Sepharose: is a tradename for a crosslinked, beaded-form of
a polysaccharide polymer material extracted from seaweed.
It is crosslinked through lysine side chains.
Sephacel: bead form of cellulose
Sephadex: crosslinked dextran/bead form of dextran
Packed (poured) into the column as a slurry

IEC Groups
Anion exchange chromatography beads (matrix/resins)
(Sulfate derivative)

Carboxymethyl (CM)

Quaternary amine

Diethylaminoethyl (DEAE)

Strong Anion Exchanger


Charged (ionized) across a
wide range of pH levels
Permanantly charged

Weak Anion Exchanger


Charged within a
narrower pH range.
Charge dependent on pH
Above 9.0 not good

Commonly Used Anion-Exchange Matrices for Protein Seperation

Cellulose Matrix
Aminoethyl-Cellulose

Exchange Type
Weak

Diethylaminoethyl (DEAE)-Cellulose

Weak

Benzyl DEAE-Cellulose

Weak

Polyethylenimine (PEI)-Cellulose

Weak

Diethyl-[2-hydroxypropyl]-aminoehtyl
(DAE)-Cellulose

Strong

Triethylaminoethyl (TEAE)-Cellulose

Weak

Diethylaminoethyl (DEAE)-Sephacel

Weak

Commonly Used Anion-Exchange Matrices for Protein Seperation


Polystyrene Matrix
Analytical Grade (AG)-1
Analytical Grade (AG)-2
Bio-Rex 5
AG 3-X4A
Bio-Rex MSZ 1-X8

Exchange Type
Strong
Strong
Intermediate
Strong
Strong

Sephadex Matrix
Diethylaminoethyl (DEAE)-Sephadex
Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)Sephadex

Exchange Type
Weak

Sepharose Matrix
Diethylaminoethyl (DEAE)-Sepharose CL-6B
Diethylaminoethyl (DEAE)-Sepharose (fast flow)
Quaternary amine (Q)-Sepharose (fast flow)

Strong
Exchange Type
Weak
Weak
Strong

Factors considered for choosing IEC matrix


(1) pH Range Where the Protein of Interest is Stable.
If the protein is most stable at pH's above its pI, then an anion-exchange gel matrix should be used. If
it is most stable below its pI, then a cation-exchange gel matrix should be used.
(2) Molecular Size of the Protein being Separated.
The porosity of an ion-exchange matrix affects the binding capacity of a gel matrix. For proteins of
molecular weight 10,000 to 100,000, DEAE-Sephacel and DEAE-Sepharose are good choices. For larger
proteins, Sephadex A-25 or C-25, which have the highest charge density at the surface of the gel, are
appropriate.
(3) Operating Pressue.
For nonrigid gels (e.g., Sephacel or Sepharose), operating pressures should not exceed the
manufacturer defined limits. For rigid gels (e.g., Analytical grade (AG), Bio-Rex MSZ, and Dowex
commercial grade ion-exchange resins from Bio-Rad), packing can be carried out at higher flow rates
using a peristaltic pump.
(4) Choice of Column Size depends on the Binding Capacity of the Ion-Exchange Gel Matrix.
The column diameters most frequently used are 1, 2, and 2.5 cm. Reservoir design, direction of
column flow, and whether or not a peristaltic pump should be employed are arbitrary.
Separating columns are usually <20 cm in length. If the protein mixture is exceedingly complex, a
longer column should be used.

Mobile Counter Ions in Anion exchange


Ions that move;
Added in buffer solution
Flow through the column
Interact with charged groups on the beads
Increasing concentration of NaCl added
Cl- is exchanged with protein

Steps of
Anion Exchange chromatography (AEC)
I. Set up column; other equipment
II. Choose buffer (pH important, above protein pI for AEC)
III. Prepare resin; pour column; equilibrate resin with buffer
IV.Load sample onto column --adsorption
V. Wash unbound proteins off the column
VI. UV spectrophotometer= OD 280 nm
VII.Desorption; elution (step gradient/continous gradient) (Collect
fractions of 1ml or 5 ml or 10ml using fraction collector)
VIII.End desorption; regenerate column (optional)

Analysis of elution profile (chromatogram)

Analyze sample for purity on SDS-PAGE


Pool fractions containing pure samples

Chromatogram

What MakesProteins Adsorb


And Desorb From The Column?
Proteins have charges because their
amino acids are charged
At a given pH, different proteins have
different charges, because they are
composed of different amino acids

Charge On Proteins
Overall net charge on a protein is
determined by the R groups of its
amino acids
Some amino acids have R group with
positive charge, others negative charge,
others no charge

In theory, IEC can separate proteins


that differ by only one charged group
Or
Two protein both negatively charged
will bind anion exchange matrix
!!!!!!!!!!
Can both be separated?

pH Of Environment
Is Crucial
pH affects stability of proteins
pH of environment affects charge on a
protein
pH is controlled with the buffer chosen

pI Is Important
First, pI for a protein of interest
determines whether use cation or an
anion resin

pI
Also, usually dont want to use a pH
where the protein has no charge at all
because it will never stick to the
beads
So, for example, pI is 6.2, may want to
use a buffer at 7.2.

IEC Principles

Order of elution: +vely will not bind and negatively charged protein will bind

IEC Principles
Mehods of Elution / desorption of
proteins bound to anion exchange
With Salt Gradient:
Counter anion Chloride ions (NaCl)
Increasing concentration
First least negatively charged proteins will elute
Followed by more negaitvely charged proteins
OR
With pH gradient
Low the pH slowly
Proteins will slowly become less negative and than
positive as pH will reach below its pI

Cation exchange chromatography


(CEC)

Cation exchange chromatography (CEC)

Beads are -vely charged


Eg Carboxymethyl (CM) matrix
+vely charged proteins bind to beads
-vely charged proteins will elute out
Add buffer to elute unbound proteins
Bound protein are eluted by changing pH or by passing a
gradually increasing linear salt gradient (NaCl high
concentration)
Generally used for basic proteins
pH of mobile phase/buffer used is 4 - 7
Basic proteins have high pI
Why for basic? If you want to use it for acidic .very low pH
may denature protein.lose its activity

Matrix or Resin
Matrix Is Support
Covalently attached functional group (charged)
Matrix, polymeric, porous beads
Cellulose
Sepharose: is a tradename for a crosslinked, beaded-form of
a polysaccharide polymer material extracted from seaweed.
It is crosslinked through lysine side chains.
Sephacel: bead form of cellulose
Sephadex: crosslinked dextran/bead form of dextran
Packed (poured) into the column as a slurry

CEC Groups
Cation exchange chromatography beads (matrix/resins)

(Sulfate derivative)

Strong Cation Exchanger


Charged (ionized) across a
wide range of pH levels
Permanently charged

Carboxymethyl (CM)

Weak Cation Exchanger


Charged within a
narrower pH range.
Charge dependent on pH
below 4.0 not good

CEC Groups
Cation

EC:negative matrix

CM = carboxymethyl celloluse/sephadex
SP = sulphopropyl
S = sulphonate

Commonly Used cation-Exchange Matrices for Protein Separation

Sephadex Matrix
Carboxymethyl (CM)-Sephadex
Sulphopropyl (SP)-Sephadex

Exchange Type
Weak
Strong

Cellulose Matrix
Carboxymethyl (CM)-Cellulose

Exchange Type
Weak

Polystyrene Matrix
AG 50W (sulphonated)
Bio-Rex 70 (carboxylic acid)

Exchange Type
Strong
Weak

Sepharose Matrix
Carboxymethyl (CM)-Sepharose CL-6B
Carboxymethyl (CM)-Sepharose (fast flow)
Sulphonate (S)-Sepharose (fast flow)

Exchange Type
Weak
Weak
Strong

Mobile Counter Ions in cation exchange


Ions that move;
Added in buffer solution
Flow through the column
Interact with charged groups on the beads
Increasing concentration of NaCl added
Na+ is exchanged with protein

Steps of
Cation Exchange chromatography (CEC)
I. Set up column; other equipment
II. Choose buffer (pH important, below protein pI for CEC)
III. Prepare resin; pour column; equilibrate resin with buffer
IV.Load sample onto column --adsorption
V. Wash unbound proteins off the column
VI. UV spectrophotometer= OD 280 nm
VII.Desorption; elution (step gradient/continous gradient) (Collect
fractions of 1 ml or 5 ml or 10ml using fraction collector)
VIII.End desorption; regenerate column (optional)

Analysis of elution profile (chromatogram)

Analyze sample for purity on SDS-PAGE


Pool fractions containing pure samples

Chromatogram
Elution profile showing absorbance at 280 on y axis and
fraction number on x axis

Charge On Proteins that bind CEC


Overall net charge on a protein is
determined by the R groups of its
amino acids
Basic protein + amino acids have R group
with more positive charge and less
negative charge
pI is generally above 7
pH range used for CEC 4-7
Protein will be +vely charged

In theory, IEC can separate proteins that differ


by only one charged group
Or
Two protein both +vely charged will bind
cation exchange matrix !!!!!!!!!!
Can both be separated? Yes.
With different salt (NaCl) conc. in elution
buffer? How?

pH Of Environment
Is Crucial
pH affects stability of proteins
pH of environment affects charge on a
protein
pH is controlled with the buffer chosen

pI
Also, usually dont want to use a pH
where the protein has no charge at all
because it will never stick to the
beads
So, for example, pI is 8.0, may want to
use a buffer at 6.8 for cation
exchange?.

IEC Principles Cation exchange

Order of elution: -vely will not bind and +vely charged protein will bind

IEC Principles
Mehods of Elution / desorption of
proteins bound to anion exchange
With Salt Gradient:
Counter anion Sodium ions (NaCl)
Increasing concentration
First least +vely charged proteins will elute
Followed by more +vely charged proteins
OR
With pH gradient
Increase the pH of buffer slowly
Proteins will slowly become less positive and than -ve
as pH will reach above its pI and elute

Protein purification system (AKTA purifier)


FPLC (fast protein liquid chromatography)

CEC chromatogram (pH increase leading to elution)

Affinity Chromatography
Adsorptive separation in which the molecule
to be purified specifically and reversibly binds
(adsorbs) to a specific chemical group (a
ligand/prosthetic
group/substrate/product/analog of
substarte/product) immobilized on an
insoluble support (a matrix or resin)
Purification is 1000X or better from a single
step (highest of all methods)
Preferred method if possible

Ligand/prosthetic group
A cofactor is a non-protein chemical compound that is bound to a protein and is
required for the protein's biological activity. These proteins are commonly
enzymes, and cofactors can be considered "helper molecules" that assist in
biochemical transformations.
Eg: Metal ions,
Vitamins
Hemoglobin (Heme + Fe)
Cofactors are either organic or inorganic.
Loosely-bound cofactors termed coenzymes
Tightly-bound cofactors termed prosthetic groups

Affinity Chromatography
X

Step 1: Attach ligand X


to column matrix

Step 2: Load protein


mixture onto column

Affinity Chromatography

Step 4: Wash column


to remove unwanted
Step 3: A specific protein
Material.
binds

to the ligand

Step 5. Elute bound protein later by providing ligand


in free form in buffer (or by changing pH or Salt
concentration)
Change in pH and salt concentration breaks reversible
interaction between protein and ligand

Affinity Chromatography
Used in many applications
Purification of substances from complex
biological mixtures
Separation of native from denatured forms of
proteins (why)
Removal of small amounts of biomaterial from
large amounts of contaminants

Affinity Chromatography
The ligand must be readily (and cheaply) available
Ligand must be attachable (covalently) to the
matrix (typically sepharose) such that it still
retains affinity for protein
Binding must not be too strong or weak
Elution involves passage of high salt or low pH
buffer after binding or unbound ligand in buffer

Example of affinity puriifcation


Lectin proteins bind specifically to sugar
molecules
Present in seeds of various plants
Ligand Glucose
Protein eluted by addition of concentrated
solution of glucose
Glucose in solution displaces column attached
glucose molecule from binding site in protein.

Affinity chromatography

Enzyme-substrate binding

Antibody-antigen binding

Immunoaffinity Chromatography
A type of Affinity chromatography
Uses antibodies
Antibodies bind very specifically to antigens.
Antigen is injected in organism (Horse/rabbit/mouse)
Antibodies are made and present in blood
Ab are purified from blood.
Purified Ab are conjugated to beads
Sample containing the antigen is passed through the affinity
column
Antigen will bind to antibodies conjugated to beads.
Elution of bound protein is done by lowering the pH to
3.0.
.

Immunoaffinity Chromatography
Antigen and Antibody interaction
Complementary shape and reversible interaction

Immunoaffinity Chromatography
Uses Immunoaffinity columns (IACs)
Small molecules can also be purified

Disadvantage: column can not be reused, antibody is denatured due to low pH

Affinity Chromatography using Protein Affinity Tags

Protein tags are peptide sequences/protein genetically fused


with a recombinant protein.
Often these tags are removable by chemical agents or by
protease enzymatic means.
Affinity tags are attached to proteins so that they can be easily
purified in one step using affinity chromatography technique.
Most common affinity tags:
Poly-His tag
Glutathione-S-transferase (GST)
Maltose binding protein (MBP)
Chitin binding protein (CBP)

Protein Affinity Tags

Immobilized metal ion affinity chromatography (IMAC)


Ni affinity chromatography
.

Allows proteins with an affinity for metal ions to be retained in a


column containing immobilized metal ions, such as cobalt, nickel,
copper for the purification of histidine tag containing proteins or
peptides

Chelator

Elution of Histag protein bound to Ni column


1. By decreasing the pH
2. By running linear gradient of imidazole

Histidine

Imidazole

Steps of Ni ion affinity chromatography

Glutathione S-transferase (GST) tags


GST is a 35-KDa protein,

GST has an affinity for glutathione


Available immobilized as glutathione agarose.

An excess amount of gluthione is used to displace the tagged protein


for elution.
The glutathione resins selectively bind to GST-tagged proteins
effectively, allowing the specific protien of interest be separated from
the mixture at high efficiency.

Step of GST-tagged protein


purification by affinity
chromatography

MBP-Tag

Maltose binding protein (MBP) tag


Maltose in buffer

Disaccharide of two glucose

Ligand

Specificity

AMP

Enzymes with NAD cofactors an ATP


dependent kinases

Arginine

Proteases such as prothrombin,


kallikrein, clostripain
Cibacron Blue Serum Albumin, Preablumin
Dye
Heparin
Growth factors, cytokines, coagulation
factors
Protein A
Fc region of immunoglobulins
Calmodulin
EGTA-copper

Calmodulin regulated kinases, cylcases


and phosphatases
Proteins with poly-Histidine tails

Size Exclusion Chromatography.


Molecules are separated according to differences
in their size as they pass through the gel-filtration
matrix
Non-adsorption technique
Ideally no interaction between matrix and
molecules
Polymer beads composed of cross-linked dextran
(dextrose) which is highly porous

SEC
Molecules with diameter greater than the largest
pores within resin material are unable to enter the
particle. Therefore they pass through the smallest
accessible volume, they travel through the column
from top to bottom fastest and elute first
Smaller molecules enter pores and access pores
within the resin particles. Pass through the larger
accessible volume within the column and beads.
Therefore elute later.
Elution is in order of decreasing molecular weight.

Size Exclusion Chromatography (SEC)

SEC

Sephadex Structure
Sephadex is a trademark for cross-linked dextran gel used for gel filtration
Dextran is a complex, branched glucan
(polysaccharide made of many glucose molecules)

SEC/ Gel-filtration chromatography


Determination of protein molecular weight
Load and run molecular weight markers
Draw standard curve
Load and run protein sample and determine elution volume

There is an inverse logarithmic relationship between the size of the molecule and the
volume eluted.

Application of SEC
Protein separation and purification
Molecular weight determination
Determination of Oligomeric state
(dimer/trimer etc)
Protein-Protein interaction

Excercise
10, 20, 40, 50 kDa protein
Which will elute first in gel filtration?
Which will elute last in gel filtration?

HIC (Hydrophobic interaction


chromatography)
Principle
Separation of substances is based on their hydrophobic
interaction with hydrophobic groups attached to an
uncharged gel matrix
Hydrophobic groups on proteins are sufficiently exposed to
bind to the hydrophobic groups on the matrix.
How is this achieved?

HIC
General concept
Salt-promoted adsorption
H2O
Protein

Hydrophobic groups on gel matrix and soluble proteins are shielded by


water molecules.
To expose these hydrophobic regions, water must be removed,
this can be achieved by adding high salt concentration in buffer
(ammonium sulfate)

HIC
H2O
Protein

Protein in buffer containing High salt


concentration (Hydrophobic patches
on protein exposed)
H2O
Protein

H2O
Protein

To elute bound protein= buffer


containing no or low salt
concentration or water only
(Hydrophobic patches get buried
inside protein)
H2O
Protein

HIC
Similar to ion-exchange chromatography except that column
material contains aromatic or aliphatic alkyl groups (phenyl,
Butyl, Octyl)
Protein attach in the presence of high salt concentration
(1M ammonium sulfate/ 2M NaCl)
Bound proteins elute using buffer containing low salt
concentration or no salt

Salting out
Salting out is a method of separating proteins based on the principle that
proteins are less soluble at high salt concentrations.
The salt concentration needed for the protein to precipitate out of the
solution differs from protein to protein.
This process is also used to concentrate dilute solutions of proteins.
Dialysis can be used to remove the salt if needed.

Principle of salting out


There are hydrophobic amino acids and hydrophilic amino acids in protein
molecules.
After protein folding in aqueous solution, hydrophobic amino acids usually
form protected hydrophobic areas while hydrophilic amino acids interact
with the molecules of solvation and allow proteins to form hydrogen
bonds with the surrounding water molecules.
If enough of the protein surface is hydrophilic, the protein can be
dissolved in water.
When the salt concentration is increased, some of the water molecules
are attracted by the salt ions, which decreases the number of water
molecules available to interact with the charged part of the protein.
As a result of the increased demand for solvent molecules, the proteinprotein interactions are stronger than the solvent-solute interactions; the
protein molecules coagulate by forming hydrophobic interactions with
each other. This process is known as salting out.
As different proteins have different compositions of amino acids, different
protein molecules precipitate at different concentrations of salt solution.

Ammonium sulfate precipitation

What follows salting out step?


Need to remove salt
Following a salting-out step, the solution will
contain a high concentration of salt that can
be disruptive to subsequent chromatographic
steps.
Dialysis

Dialysis
Movement of molecules by diffusion from high
concentration to low concentration through a
semi-permeable membrane.
Only those molecules that are small enough to fit through the
membrane pores are able move through the membrane and
reach equilibrium with the entire volume of solution in the
system
By contrast, large molecules that cannot pass through the
membrane pores will remain on the same side of the
membrane as they were when dialysis was initiated.
To remove additional unwanted substance, it is necessary to
replace the dialysis buffer so that a new concentration
gradient can be established.
Once the buffer is changed, movement of particles from high
(inside the membrane) to low (outside the membrane)
concentration will resume until equilibrium is once again
reached.

Separation based on SIZE


Dialysis:
for separating proteins from small molecules & ions
Small molecules (blue) (buffer ions, small organic molecules, salt etc.)
pass through membrane, diffusing into surrounding medium.
Dialysate (buffer in which dialysis bag is suspended) can be changed
frequently --> serial dilution, getting rid of unwanted small molecules
and ions.
Dialysis membranes with different pore sizes available

Factors Affecting Dialysis Rate


Factors that affect the completeness of dialysis include
(1) dialysis buffer volume,
(2) buffer composition,
(3) the number of buffer changes,
(4) time,
(5) temperature and
(6) particle size vs. membrane pore size.